Chimeric antigen receptor to which anti-cotinine antibody is linked, and use thereof

ABSTRACT

A chimeric antibody receptors with anti-cotinine antibodies linked, and uses thereof are disclosed. A T cell presenting the chimeric antibody receptor on the surface secretes interferon gamma specifically for a target molecule of a cotinine-conjugated binding molecule that is added together therewith and induces cell death of the cell expressing the target molecule by the T cell. On the contrary, by administering a cytotoxic agent conjugated with cotinine, cell death of the chimeric antigen receptor T cell is induced. Therefore, if necessary, a cytotoxic agent conjugated with cotinine can be administered to remove the chimeric antigen receptor T cells that have been already administered, thereby suppressing immune side effects due to hyperactivity of T cells. Thus, the chimeric antigen receptor to which the anti-cotinine antibody is linked can be effectively and safely used for the treatment of cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No. 15/753,212filed Feb. 16, 2018 (allowed), which is a National Stage ofInternational Application No. PCT/KR2016/009047 filed Aug. 17, 2016,claiming priority based on U.S. Provisional Patent Application No.62/205,844 filed Aug. 17, 2015.

SEQUENCE LISTING

The content of the electronically submitted sequence listing, file name:Sequence_Listing_As_Filed.xml; size: 53,811 bytes; and date of creation:Aug. 26, 2022, filed herewith, is incorporated herein by reference inits entirety.

TECHNICAL FIELD

The present invention relates to a chimeric antigen receptor linked withan anti-cotinine antibody and a use thereof.

BACKGROUND ART

While substances such as peptides, aptamers, antibodies, etc. that canbe developed as biological therapeutics have excellent effects, suchsubstances can be easily broken down in the body and rapidly releasedthrough the kidneys, and thus have short in vivo half-lives. In order toresolve such problem, studies have been conducted to increase the invivo half-lives of the substances by conjugating polyethylene glycol(PEG) thereto (Veronese F. M. & Pasut G., Drug Discov. Today, 10,1451-1458, 2005). However, in the process of conjugating PEG, variousmolecular complexes are formed, and there is a limitation thatoptimization conditions must be established depending on bindingmolecules which are conjugated to PEG. In addition, such molecules havethe disadvantage of having no or weak cytotoxicity when the targetobject thereof is a cell (e.g., a cancer cell, etc.). Therefore,recently, conjugates of an antibody and a cytotoxic agent (antibody-drugconjugates) are being developed.

Meanwhile, a chimeric antigen receptor (CAR) is composed of a portion ofan antibody, a hinge region, a transmembrane domain, and anintercellular signal transduction domain. Immune cells such as T cellsand natural killer cells expressing the CAR (CAR cells) specificallyrecognize a surface molecule that expresses on the target cells (e.g.,cancer cells, etc.) using an antibody portion of the CAR, and showcytotoxicity to the target cells. Therefore, the CAR cells are utilizedas a form of genetically engineered cell therapy, and in particular, itwas reported that T cells expressing CAR (CAR T cells) showed a hightherapeutic effect for hematological malignancies in which CD19 areexpressed.

However, since conventional CAR cells have a limitation of recognizingonly a single target molecule, CAR cells against haptens have beenstudied. These cells express CARs that recognize a specific hapten usingan antibody portion of the CAR. Then, the molecules, such as peptides,aptamers, antibodies, low molecular weight chemicals, etc., that possessthe binding capacity to a target molecule, are conjugated with thehapten and complexed with these anti-hapten CAR cells. Thereby,multi-targeting CAR cells can be prepared, in which one type of CARcells can bind to various target molecules.

In order to prevent cells expressing chimeric antigen receptor frombinding with non-target cells, it is preferable that the hapten be asubstance which does not originally exist in vivo. In addition, thehapten must have a chemical functional group for effective conjugationwith a binding molecule.

DISCLOSURE OF INVENTION Technical Problem

Cotinine, a major metabolite of nicotine, is not biosynthesized in vivo(de novo biosynthesis) and does not show physiological activity. Inaddition, a metabolic process of cotinine in mammals has been wellknown, and it has also been reported that the serum half-life ofcotinine is as short as 16 hours (Benowits N. L. et al., 3rd Handb. Exp.Pharmacol., 29-60, 2009). Therefore, even in the case of smokers,cotinine can be used as a hapten because cotinine is no longer presentin the body after smoking has been stopped for several days. Inaddition, since cotinine with carboxyl functional group is easilyavailable, cotinine conjugation to a binding molecule can befacilitated.

As such, by preparing T cells expressing CAR harboring a portion of ananti-cotinine antibody and a cotinine-conjugated binding molecule, thepresent inventors confirmed that the CAR T cells recognize the target ofthe cotinine-conjugated binding molecule specifically, secreteinterferon gamma, and induce cell death of target cells (FIG. 1 ).

In addition, recently, along with the increased efficacy of anti-tumorCAR T cell therapy, immunotoxicity due to the overactivity of suchgenetically engineered T cells is being highlighted as an adverse sideeffect. Therefore, measures are being investigated for resolving seriousadverse side effects caused by these T cells by inducing cell death ofsuch cells when the adverse side effects develop. In the case of theanti-hapten CARs, when an immunotoxic side effect occurs in the body ofa patient injected with the CAR T cells, injecting a hapten-conjugatedcytotoxic agent instead of a hapten-conjugated binding molecule caninduce cell death of the CART cells because the hapten-conjugatedcytotoxic agent binds only to the anti-hapten CART cells. Thus, thissystem provides an additional advantage that the hapten-conjugatedcytotoxic agent can be used as a safety measure to resolve adverse sideeffects caused by the T cells (FIG. 2 ). Accordingly, the presentinventors accomplished the present invention by confirming that celldeath of the chimeric antigen receptor T cells is induced by acotinine-conjugated cytotoxic agent.

Therefore, it is an object of the present invention to provide a CARlinked with an anti-cotinine antibody.

It is another object of the present invention to provide a nucleic acidmolecule encoding the CAR, an expression vector and a virus includingthe same, and a cell transduced with the virus.

It is still another object of the present invention to provide a CARcell further comprising a cotinine-conjugated binding molecule in thecell.

It is still another object of the present invention to provide apharmaceutical composition for preventing or treating a condition or adisease in which specific cells are proliferated, the pharmaceuticalcomposition comprising the CAR cell as an active ingredient.

It is still another object of the present invention to provide a methodfor preventing or treating a condition or a disease in which specificcells are proliferated, the method comprising administering the CAR cellto a subject.

It is still another object of the present invention to provide a methodfor inducing cell death of the CAR cell.

Solution to Problem

In order to achieve the above objects, the present invention provides aCAR linked with an anti-cotinine antibody, the CAR comprising ananti-cotinine antibody or a fragment thereof, a hinge domain, atransmembrane domain, and a signal transduction domain.

In addition, the present invention provides a nucleic acid moleculeencoding the CAR.

In addition, the present invention provides an expression vectorcomprising the nucleic acid molecule.

In addition, the present invention provides a virus comprising thenucleic acid molecule.

In addition, the present invention provides a cell transduced with thevirus.

In addition, the present invention provides a CAR cell comprising acotinine-conjugated binding molecule in the cell.

In addition, the present invention provides a pharmaceutical compositionfor preventing or treating a condition or a disease in which specificcells are proliferated, the pharmaceutical composition comprising theCAR cell as an active ingredient.

In addition, the present invention provides a method for preventing ortreating a condition or a disease in which specific cells areproliferated, the method comprising administering the CAR cell to asubject.

In addition, the present invention provides a method for preparing a CARcell in a subject, the method comprising administering the cell to thesubject; and administering a cotinine-conjugated binding molecule to thesubject.

In addition, the present invention provides a method for inducing celldeath of a CAR cell, the method comprising administering the cell and acotinine-conjugated binding molecule to a subject; and furtheradministering a cotinine-conjugated cytotoxic agent to the subject.

Furthermore, the present invention provides a pharmaceutical kit forpreventing or treating a condition or a disease in which specific cellsare proliferated, the pharmaceutical kit comprising a first componentcontaining the CAR cell as an active ingredient in a pharmaceuticallyand therapeutically effective amount; and

a second component containing a complex of cotinine and a cytotoxicagent an as an active ingredient in a pharmaceutically andtherapeutically effective amount.

Advantageous Effects of Invention

T cells, which present, on the surface thereof, the CAR linked with ananti-cotinine antibody according to the present invention, recognize thetarget of the cotinine-conjugated binding molecule specifically, secreteinterferon gamma, and induce cell death of cells expressing the targetmolecule. In addition, by administering a cotinine-conjugated cytotoxicagent, cell death of the CART cells is induced. Accordingly, when it isnecessary, it is possible to remove pre-administered CAR T cells byadministering a cotinine-conjugated cytotoxic agent, thereby suppressadverse immune side effects due to overactivity of T cells. Therefore,the chimeric antigen receptor linked with an anti-cotinine antibody canbe effectively used to treat a condition or a disease in which specificcells are proliferated.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing that the target of a chimeric antigenreceptor T cell can be switched by changing a cotinine-conjugatedbinding molecule depending on a target cell.

FIG. 2 is a diagram showing that a cotinine-conjugated cytotoxic agentis used to induce cell death of chimeric antigen receptor T cells.

FIGS. 3A and 3B show the structure of a chimeric antigen receptor genelinked with an anti-cotinine antibody fragment (Cot-28Z) and a virusvector expressing the same prepared according to an embodiment of thepresent invention (pMP-Cot-28Z), and a graph of the results of analyzingT cells which present the chimeric antigen receptor on the surfacethereof by a flow cytometer, respectively.

FIG. 4 is a graph showing the amounts of interferon gamma secreted by Tcells, when chimeric antigen receptor T cells linked with theanti-cotinine antibody fragment mixed with a cotinine-conjugatedanti-VEGF aptamer (Cot-Macugen) prepared according to an embodiment ofthe present invention are reacted to a target molecule, VEGF protein.

FIG. 5 is a graph showing the amounts of interferon gamma secreted by Tcells, when the chimeric antigen receptor T cells mixed with acotinine-conjugated anti-HER2 antibody (anti-HER2-Cot) preparedaccording to an embodiment of the present invention are added to HER2positive or negative tumor cells.

FIGS. 6A and 6B are a graph confirming the HER2-positive tumor-specificinterferon gamma production of the chimeric antigen receptor T cellsmixed with a cotinine-conjugated anti-HER2 antibody (Herceptin-cot)prepared according to an embodiment of the present invention, and agraph showing the results of analyzing chimeric antigen receptorsexpressed on the surface of T cells by a flow cytometer, respectively.

FIG. 7 is a graph showing the amounts of interferon gamma secreted by Tcells, when the chimeric antigen receptor T cells mixed with acotinine-conjugated anti-CD20 antibody (anti-CD20-cot) preparedaccording to an embodiment of the present invention are added to CD20positive or negative tumor cells.

FIG. 8 is a graph showing the amounts of interferon gamma secreted by Tcells, when the chimeric antigen receptor T cells mixed with acotinine-conjugated anti-HLA antibody (anti-HLA-cot) prepared accordingto an embodiment of the present invention are added to HLA positive ornegative tumor cells.

FIG. 9A is a graph showing target cells stained to show fluorescencehaving different intensities and FIG. 9B is a graph showing the resultsof adding the chimeric antigen receptor T cells prepared and acotinine-conjugated anti-HER2 antibody according to an embodiment of thepresent invention to the target cells, and analyzing the targetcell-specific cell death effect of the chimeric antigen receptor T cellsby a flow cytometer.

FIGS. 10A-10C are graphs showing the cell death-inducing effect of thechimeric antigen receptor T cells by a cotinine-conjugated cytotoxicagent (A: cotinine-duocarmycin single conjugate, B: cotinine-DM1 complexconjugate (2×cotinine-4×DM1), and C: cotinine-duocarmycin complexconjugate (2×cotinine-4×duocarmycin)).

FIGS. 11A-11C are a set of diagrams showing the structures of cytotoxicagents used in an embodiment of the present invention (A) acotinine-duocarmycin single conjugate, (B) a cotinine-DM1 complexconjugate, and (C) a cotinine-duocarmycin complex conjugate.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

The present invention provides a chimeric antigen receptor linked withan anti-cotinine antibody, the chimeric antigen receptor comprising ananti-cotinine antibody or a fragment thereof, a hinge domain, atransmembrane domain, and a signal transduction domain.

The chimeric antigen receptor linked with the anti-cotinine antibody maybe in a form in which an anti-cotinine antibody or a fragment thereof, ahinge domain, a transmembrane domain, and a signal transduction domainare linked in this order in the direction from the N-terminus to theC-terminus.

The anti-cotinine antibody is an antibody that binds to cotinine, andthe antibody may be any one of a monoclonal antibody, a polyclonalantibody, or a recombinant antibody. In addition, the antibody may be anantibody in a full-length form or a fragment thereof. Here, the fragmentof an antibody may comprise a part of an anti-cotinine antibody capableof binding to cotinine. The fragment of an antibody may be Fab, Fab′,F(ab′)2, Fv, or scFv.

The anti-cotinine antibody or a fragment thereof may include a heavychain variable region which includes CDR1, CDR2, or CDR3, where CDR1,CDR2, and CDR3 are complementarity determining regions (CDR) representedby the amino acid sequences of SEQ ID NOs: 23 to 25, respectively, or alight chain variable region including CDR1, CDR2 or CDR3, where CDR1,CDR2, and CDR3 are represented by the amino acid sequences of SEQ IDNOs: 26 to 28, respectively. Specifically, the above-mentionedanti-cotinine antibody or a fragment thereof may include a heavy chainvariable region including CDR1, CDR2, and CDR3 represented by the aminoacid sequences of SEQ ID NOs: 23 to 25, respectively, and a light chainvariable region including CDR1, CDR2, and CDR3 represented by the aminoacid sequences of SEQ ID NOs: 26 to 28, respectively. In an embodimentof the present invention, the anti-cotinine antibody may be a scFvfragment having an amino acid sequence of SEQ ID NO: 1. In addition, theanti-cotinine antibody may be at least 80%, 85%, 90%, 95%, 97%, 98%, or99% identical to the amino acid sequence of SEQ ID NO: 1.

The anti-cotinine antibody or a fragment thereof according to thepresent invention can be prepared by modifying the method described inU.S. Pat. No. 8,008,448.

The hinge domain is a domain for connecting an anti-cotinine antibody ora fragment thereof and a transmembrane domain, and may include acysteine residue. The cysteine residue may be involved in the binding ofa hinge domain to an antibody. For example, the hinge domain may be aCD8 hinge domain, an IgG1 hinge domain, an IgG4 hinge domain, a CD28extracellular domain, a killer immunoglobulin-like receptor (KIR)extracellular domain, or a combination thereof (Mol. Ther, 2015(23):757; J. Immunother., 2006(29):284; Cancer Immunol. Res., 3(7):815-26;Blood, 2013(122):2965). The CD8 hinge domain may have an amino acidsequence of SEQ ID NO: 3. In addition, the CD8 hinge domain may be atleast 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acidsequence of SEQ ID NO: 3.

The transmembrane domain may connect the hinge domain of the chimericantigen receptor and the signal transduction domain. The transmembranedomain may penetrate cell membranes of cells such that the anti-cotinineantibody of the chimeric antigen receptor or a fragment thereof islocated on the surface of the cells and the signal transduction domainis located intracellularly. The transmembrane domain may be atransmembrane region of the CD3 zeta, CD4, CD8, CD28, or KIR protein(Immunol. Rev., 2014(257):107; J. Immunol., 2010(184):6938; Blood, 2013(122): 2965, J. Immunother, 2006(29):284; Cancer Immunol. Res.,3(7):815-26). In an embodiment of the present invention, thetransmembrane domain may include a transmembrane region and acytoplasmic region of CD28, and the domain may have an amino acidsequence of SEQ ID NO: 5 or SEQ ID NO: 7. In addition, the transmembranedomain may be at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical tothe amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7.

The signal transduction domain receives a signal provided by ananti-cotinine antibody or a fragment thereof and plays a role intransmitting the signal into the cell to which the chimeric antigenreceptor is bound. The signal transduction domain may be CD3 zeta, CD278(inducible T-cell costimulator, ICOS), CD28, CD134 (OX40), CD137(4-1BB), killer immunoglobulin-like receptor (KIR), or DNAX activationprotein 12 (DAP12) (J. Immunol., 2004(172):104; Mol. Ther,2013(21):2268; Proc. Natl. Acad Sci. USA, 2009(106):3360; CancerImmunol. Res., 3(7):815-26). Specifically, the signal transductiondomain may be a cytoplasmic region of CD28 and CD3 zeta, or acytoplasmic region of CD137 (4-1BB) and CD3 zeta.

In an embodiment of the present invention, the signal transductiondomain may be a cytoplasmic region of CD28 and CD3 zeta. The cytoplasmicregion of CD28 may have an amino acid sequence of SEQ ID NO: 9 or SEQ IDNO: 11, and the cytoplasmic region of CD3 zeta may have an amino acidsequence of SEQ ID NO: 13. In addition, the signal transduction domainmay be at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to theamino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

Furthermore, the chimeric antigen receptor of the present invention mayinclude a modified form of the antibody and the domain as describedabove. Here, the modification may be carried out by substituting,deleting, or adding one or more amino acids of the amino acid sequenceof a wild-type antibody and domain without modifying the function of theantibody and the domain. Conventionally, the substitution may be alanineor may be carried out by conservative amino acid substitution which doesnot affect the charge, polarity, or hydrophobicity of the whole protein.In an embodiment of the present invention, the chimeric antigen receptormay have an amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 17. Inthe sequence of SEQ ID NO: 15, amino acid residues at positions 22-266correspond to scFv (SEQ ID NO: 1), amino acid residues at positions286-348 correspond to CD8 hinge (SEQ ID NO: 3), amino acid residues atpositions 349-376 correspond to human CD28 transmembrane domain (SEQ IDNO: 5), and amino acid residues at positions 377-417 and 420-531 eachcorrespond to human CD28 cyt (SEQ ID NO: 9) and human CD3 zeta cyt (SEQID NO: 13), respectively. In the sequence of SEQ ID NO: 17, amino acidresidues at positions 22-266 correspond to scFv (SEQ ID NO: 1), aminoacid residues at positions 286-348 correspond to CD8 hinge (SEQ ID NO:3), amino acid residues at positions 349-378 correspond to mouse CD28transmembrane domain (SEQ ID NO: 7), and amino acid residues atpositions 379-419 and 422-533 each correspond to mouse CD28 cyt (SEQ IDNO: 11) and CD3 zeta cyt (SEQ ID NO: 13), respectively. Human CD8 hingeand mouse CD8 hinge share the same sequence of SEQ ID NO: 3, and humanCD3 zeta cyt and mouse CD3 zeta cyt share the same sequence of SEQ IDNO: 13. In addition, the chimeric antigen receptor may be at least 80%,85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO: 15 or SEQ ID NO: 17.

In addition, the present invention provides a nucleic acid moleculeencoding the chimeric antigen receptor.

The nucleic acid molecule according to the present invention may encodethe anti-cotinine antibody or a fragment thereof (SEQ ID NO: 2), thehinge domain (SEQ ID NO: 4), the transmembrane domain (SEQ ID NO: 6 or8), and the signal transduction domain (SEQ ID NO: 10, 12, or 14)described above. Here, the nucleic acid molecule encoding the chimericantigen receptor according to the present invention may be a nucleotidesequence of SEQ ID NO: 16 or SEQ ID NO: 18. Here, the nucleotidesequence may include another substituted nucleotide sequence capable ofexpressing the antibody or domain of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. Inaddition, the nucleic acid molecule may include a nucleic acid moleculeencoding the antibody or the domain in the modified form as describedabove.

In addition, the present invention provides an expression vectorincluding the nucleic acid molecule.

The expression vector may be an adenovirus vector, a retrovirus vector,a lentiviral vector, or an adeno-associated virus vector. In anembodiment of the present invention, the expression vector may be aretrovirus vector. The expression vector may be prepared by thoseskilled in the art such that the chimeric antigen receptor according tothe present invention is expressed and secreted.

The expression vector may further include a signal sequence or a leadersequence. In an embodiment of the present invention, the leader sequencemay be an immunoglobulin kappa leader sequence. The immunoglobulin kappaleader sequence may have an amino acid sequence of SEQ ID NO: 19, andmay be at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to theamino acid sequence of SEQ ID NO: 19.

The present invention also provides a virus comprising the nucleic acidmolecule.

The virus may include, in the genome thereof, a nucleic acid moleculeencoding the chimeric antigen receptor according to the presentinvention. Therefore, a cell transduced with the virus may express thechimeric antigen receptor according to the present invention, in whichan anti-cotinine antibody or a fragment thereof is located on thesurface thereof. The virus may be an adenovirus, a retrovirus, alentivirus, or an adeno-associated virus. In an embodiment of thepresent invention, the virus may be a retrovirus.

The virus may be obtained by transfecting cells with the expressionvector as described above together with an expression vector comprisinga nucleic acid molecule encoding a viral envelope protein. Here,transfection can be carried out by a conventional method. Fortransfection, the envelope protein that can be used may be VSV-G, anecotropic envelope, Mokola, Rabies, MLV-Ampho, MLV-10A1, LCMV-WE,LCMV-Arm53b envelope, feline endogenous gamma retrovirus RD114 envelopeand a variant thereof, gibbon ape leukemia virus (GALV) envelope and avariant thereof, MLV 4070A envelope, or gp120/gp41 (Mol. Ther MethodsClin. Dev., 2016(3):16017; J. Virol. Methods, 2004:122-131; MolecularTherapy-Methods & Clinical Development 2016(3): 16017).

In addition, the present invention provides a cell transduced with thevirus as described above.

The cell may be transduced with a virus comprising a nucleic acidcapable of expressing the chimeric antigen receptor according to thepresent invention. The transduced cell may have an anti-cotinineantibody or a fragment thereof located on the surface of the cell, andwhen cotinine binds to the antibody or the fragment thereof viaantigen-antibody binding, cellular activity may be induced throughintracellular signal transduction. Here, the cell may be a T cell, anatural killer cell, or a macrophage. In an embodiment of the presentinvention, the cell may be a T cell.

In addition, the present invention provides a chimeric antigen receptorcell further comprising a binding molecule to which cotinine isconjugated.

In the cotinine-conjugated binding molecule, when the binding moleculeis an antibody, the complex may be formed via a bond between thecarboxyl group of cotinine and the amine group of the antibody.

The cotinine, being a hapten, may bind to an antibody that specificallyrecognizes cotinine without changing the properties of thecotinine-conjugated binding molecule. Specifically, the binding moleculemay be a peptide, a nucleic acid, a protein, or a chemical substance.The nucleic acid may be an aptamer, and the protein may be an antibodyor a hormone. In an embodiment of the present invention, the aptamer maybe an anti-VEGF aptamer. In addition, in an embodiment of the presentinvention, the antibody may be an anti-HER2 antibody, an anti-CD20antibody, or an anti-HLA antibody.

The chimeric antigen receptor cell may harbor the anti-cotinine antibodyor the fragment thereof linked to the cell. When the cotinine conjugatesbind to an anti-cotinine antibody or a fragment thereof, they may inducecellular activities. Here, the binding of the cell andcotinine-conjugated binding molecule may be an antigen-antibody bindingbetween the anti-cotinine antibody of the chimeric antigen receptor or afragment thereof and cotinine. In addition, the cellular activity may becell death-inducing activity by cytotoxic T cells.

The chimeric antigen receptor cell may be prepared through a step ofadding a cotinine-conjugated binding molecule to the cell in which achimeric antigen receptor linked with an anti-cotinine antibody or afragment thereof expresses, and a step of selecting a chimeric antigenreceptor cell complexed with the binding molecule.

In addition, the present invention provides a pharmaceutical compositionfor preventing or treating a condition or a disease in which specificcells are proliferated, comprising the chimeric antigen receptor cell asan active ingredient.

The chimeric antigen receptor cell used as the active ingredient of thepharmaceutical composition is as described above.

The condition or disease may be cancer, and the cancer may be solidcancer or hematologic malignancy. Here, the solid cancer may be lungcancer, colon cancer, prostate cancer, thyroid cancer, breast cancer,brain cancer, head and neck cancer, esophageal cancer, skin cancer,melanoma, retinoblastoma, thymic cancer, gastric cancer, colorectalcancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer,rectal cancer, gall bladder cancer, bile duct cancer, or pancreaticcancer. In addition, the hematologic malignancy may be lymphoma,leukemia, or multiple myeloma.

The pharmaceutical composition may comprise 10 to 95 wt % of thechimeric antigen receptor cells according to the present invention as anactive ingredient based on the total weight of the pharmaceuticalcomposition. In addition to the above-mentioned active ingredient, thepharmaceutical composition of the present invention may further includeone or more types of active ingredients exhibiting the same or similarfunctions.

In addition to the active ingredients described above, thepharmaceutical composition according to the present invention mayfurther comprise one or more types of pharmaceutically acceptablecarriers for administration.

In addition, the present invention provides a method for preventing ortreating a condition or a disease in which specific cells areproliferated, the method including administering the chimeric antigenreceptor cell to a subject.

The chimeric antigen receptor cell is as described above. In addition,the condition or disease may be cancer, and specific cancers are asdescribed above.

The dosage amount of the chimeric antigen receptor cell according to thepresent invention may be adjusted according to various factors such asthe type of a disease, the severity of a disease, the type and contentof active ingredients and other ingredients included in thepharmaceutical composition, the type of dosage form and the age, bodyweight, general health, sex, and diet of a patient, administration time,administration route, treatment time, and concurrently used drugs.However, for desired effects, the effective amount of the chimericantigen receptor cells included in the pharmaceutical compositionaccording to the present invention may be 1×10⁵ to 1×10¹¹ cells/kg.Here, the administration may be performed once a day and may also bedivided over several times a day.

In addition, the chimeric antigen receptor cell of the present inventionmay be administered to a subject by various methods known in the art.The subject may be a mammal, and specifically a human. Theadministration route may be appropriately selected by those skilled inthe art in consideration of the administration method, volume andviscosity of a body fluid, and the like.

In addition, the present invention provides a method for producing achimeric antigen receptor cell in a subject, the method comprisingadministering the cell to the subject; and administering acotinine-conjugated binding molecule to the subject.

The cell is transduced with a virus comprising a nucleic acid capable ofexpressing the chimeric antigen receptor according to the presentinvention, and is as described above. In addition, thecotinine-conjugated binding molecule is as described above.

The subject may be a mammal, and specifically a human, andadministration may be appropriately carried out by those skilled in theart as described above.

In addition, the present invention provides a method for inducing celldeath of a chimeric antigen receptor cell, the method comprisingadministering the cell and a cotinine-conjugated binding molecule to asubject; and further administering a cotinine-conjugated cytotoxic agentto the subject.

The chimeric antigen receptor cell is as described above. In the case ofan anticancer treatment using chimeric antigen receptor cells,immunotoxicity due to overactivity of the cells may occur as a sideeffect. Therefore, in order to resolve such a side effect, cell death ofchimeric antigen receptor cells can be induced by additionallyadministering a cotinine-conjugated cytotoxic agent in addition to acotinine-conjugated binding molecule.

The cytotoxic agent mentioned above may be used as long as it is asubstance capable of inducing cell death. Specifically, the cytotoxicagent may be duocarmycin, SN-38, calicheamicin, monomethyl auristatin E(MMAE), monomethyl auristatin F (MMAF), doxorubicin,pyrrolobenzodiazepine, DM4, or DM1. In an embodiment of the presentinvention, the cytotoxic agent may be duocarmycin or DM1 (ASCO meetingabstracts 2014(32):2558; Clin. Cancer Res., 2011(17):3157-69; Leuk.Lymphoma, 2015(56): 2863-69; J. Clin. Oncol., 2014(32):3619-25; Proc.Am. Soc. Clin. Oncol., 2015(33):2503; J. Drug Deliv., 2013(2013):898146;Sci. Transl. Med., 2015(7):302ra136; Mol, Cancer Ther, 2014(13):1537-48; J. Clin. Oncol., 2008(26):2147-54).

Administration of the cotinine-conjugated cytotoxic agent may beadjusted by various factors as described above. The effective amount ofthe chimeric antigen receptor cells included in the pharmaceuticalcomposition according to the present invention may be appropriatelydetermined by those skilled in the art. Here, the administration may beperformed once a day and may also be divided over several times a day.

Furthermore, the present invention provides a pharmaceutical kit forpreventing or treating a condition or a disease in which specific cellsare proliferated, the pharmaceutical kit comprising a first componentcontaining the chimeric antigen receptor cell as an active ingredient ina pharmaceutically and therapeutically effective amount; and a secondcomponent containing a cotinine-conjugated cytotoxic agent as an activeingredient in a pharmaceutically and therapeutically effective amount.

In the pharmaceutical kit of the present invention, the first componentand the second component may be administered sequentially for theprevention or treatment of a condition or a disease. In the case ofsequentially administering the first component and the second component,the second component may be administered when an immunotoxic side effectappears or when the chimeric antigen receptor cells are immortalizedafter the administration of the first component.

The condition or disease may be cancer, and specific cancers are asdescribed above.

MODE FOR THE INVENTION

Hereinafter, the present invention is described in detail using theexamples below. The examples below are merely intended exemplify thepresent invention, and the scope of the present invention is not limitedthereto.

Example 1. Preparation of Retrovirus Comprising Nucleic Acid EncodingChimeric Antigen Receptor Linked with Anti-Cotinine Antibody Fragment1-1. Preparation of Construct

First, a plasmid comprising nucleic acids each encoding an scFv fragmentof an anti-cotinine antibody, a hinge domain, a transmembrane domain,and a signal transduction domain was prepared by the following method.

Specifically, a scFv fragment of an anti-cotinine antibody was amplifiedby PCR in a conventional manner from a template plasmid using a primerincluding a mouse Ig kappa leader sequence. The template was a plasmidgenerated by ligation of an anti-cotinine scFv fragment cleaved with theSfiI restriction enzyme to a pCEP4 vector (Invitrogen, US) (Clin. Chim.Acta., 2010, 411-1238). Meanwhile, the skeletal part of the chimericantigen receptor including c-myc tag(SEQ ID NO. 21), human CD8 hingeregion, transmembrane region and cytoplasmic region of mouse CD28, and acytoplasmic region of human CD3 zeta, was amplified by PCR from thetemplate, pLxSN-scFv-anti-CEA-CD28-zeta plasmid (Dr. Philip Darcy,PeterMac Cancer Center, Australia). Specific sequences of the primersused for the PCR are shown in Table 1 below.

TABLE 1 Name Sequence (5′→3′) SEQ ID Number Anti-cotininegatatcaagcttgccaccatggattttcaggtgcagattttcagcttcctgct SEQ ID NO: 29scFv forward aatcagtgcctcagtcataatgtctagagagctcgatctgacccag directionAnti-cotinine tgaagagatggtgaccag SEQ ID NO: 30 scFv reverse directionCAR skeleton gcggccgcagaacaaaaa SEQ ID NO: 31 forward directionCAR skeleton actagtgtcgacttagcgagggggcagggc SEQ ID NO: 32 forwarddirection CEA CAR gatatcaagcttccatgggccaccatggattttcaggtgcagSEQ ID NO: 33 forward direction CEA CARgaattcatcgatgtcgacgcggccgcttagcgagggggcagggc SEQ ID NO: 34 reversedirection

The two obtained PCR products were ligated to prepare cDNA of theanti-cotinine chimeric antigen receptor. The prepared cDNA and thepMSCV-puro vector (K1062-1, Clontech, US) were cleaved with HindIII/SalIand HindIII/ClaI, respectively and ligated together. As a result, thecDNA encoding the scFv fragment of the anti-cotinine antibody and theskeletal domains of chimeric antigen receptor was cloned into theposition downstream of the PGK promoter of a pMSCV-puro retroviralvector, where the puromycin-resistant gene had been originally located.The prepared construct was confirmed through nucleotide sequencinganalysis, and named pMP-Cot-28Z (FIG. 3A).

1-2. Preparation of Retrovirus

The retroviral construct prepared in Example 1-1 was transfected intoPHOENIX™ GP (ATCC, US) cell line, along with pMD2.G plasmid (#12259,Addgene, US) which contains cDNA encoding the vesicular stomatitisIndiana virus G protein (VSV-G) as a virus envelope protein.Transfection was carried out according to the manufacturer's protocolusing LIPOFECTAMINE™ 2000 (Cat. #11668-019, Invitrogen, US). After 48hours, the culture supernatant containing the VSV-G pseudotypedretrovirus was harvested and incubated with a PHOENIX™ Eco (ATCC, US)cell line for infection with the retrovirus. Three to five days afterinfection, Phoenix Eco cells positively stained withfluorescence-labeled anti-myc antibody were selected using a flowcytometer (BD Bioscience, US) to establish a retrovirus-producing cellline. The retrovirus culture supernatant produced from this cell linewas concentrated 10-fold using a centrifugal filter device (AmiconUltra-15, 100 kDa cut-off, Millipore, US).

Example 2. Preparation of Cytotoxic T Cells with a Chimeric AntigenReceptor Linked with Anti-Cotinine Antibody Fragment Presented onSurface Thereof

Using the virus prepared in Example 1-2, cytotoxic T cells were preparedwith a chimeric antigen receptor linked with anti-cotinine antibodyfragment presented on the surface thereof.

First, splenocytes were isolated from B6 mice and dispensed in a cellculture container coated with 10 μg/mL of the anti-CD3 antibody(145-2C11, BD Bioscience, US) so that there were 2.5×10⁶ cells per well.The cells were supplemented with a total of 1 mL of the RPMI-1640 mediumincluding 2 μg/mL of the anti-CD28 antibody (37.51, BD Bioscience, US),and cultured under conditions of 5% CO² and 37° C. After 24 hours, 1 mLof the concentrated retrovirus including 2 μL of polybrene(Sigma-Aldrich, US) at 6 mg/mL was added to the cells and transduced bycentrifugation for 90 minutes at 2,500 rpm using a centrifuge(Centrifuge 5810R, Eppendorf, US). Polybrene was used to increase thetransduction rate of the retrovirus. Afterwards, 1 mL of the culturesupernatant was removed, 1 mL of the concentrated retrovirus and 1 μL ofpolybrene were added thereto, and the retrovirus was transduced bycentrifugation under the same conditions as above. Afterwards, 1 mL ofthe culture solution was removed, and 1 mL of the RPMI-1640 mediumincluding 20 U/mL of interleukin-2 (Gibco, US) was added.

After 48 hours, the medium of the virus-transduced T cells was replacedwith a medium containing 20 U/mL of interleukin-2 and cultured for 72hours under conditions of 5% CO² and 37° C. Afterwards, the transductionrate of the chimeric antigen receptor linked with the anti-cotinineantibody fragment was determined by measuring the percentage of cellpopulation positively stained with fluorescence-labeled anti-c-mycantibody using a flow cytometer, which was about 50 to 70% (FIG. 3B).Meanwhile, the T cells in which the green fluorescent protein wasintroduced as a control group showed a transduction rate of about 80%. Tcells with the thus prepared chimeric antigen receptor linked with theanti-cotinine antibody fragment presented on the surface thereof wereused in the following experiments within 24 hours after preparation.

Example 3. Preparation of Complex of Cotinine and Binding Molecule 3-1.Preparation of Complex of Cotinine and Antibody

As a binding molecule, cotinine-conjugates were prepared using rituximab(Genentech, Biogen, US), which is an anti-CD20 antibody, trastuzumab(Genentech, US), which is an anti-HER2 antibody, CT302 (Celltrion,Korea), which is an anti-influenza virus hemagglutinin antibody, andW6/32 (eBioscience, US), which is an anti-HLA antibody. Cotinineconjugation was performed by an EDC (1-ethyl-3- [3-dimethylaminopropyl]carbodiimide) coupling method.

First, the antibodies above were dissolved in PBS to a concentration of25 μM. Meanwhile, trans-4-cotinine carboxylic acid (Sigma-Aldrich) wasdissolved in 1 mL of the MES buffer [0.1 M MES(2-[morpholino]ethanesulfonic acid) and 0.5 M sodium chloride, pH 6.0]to a concentration of 5 mM. EDC at a concentration of 50 mM andN-hydroxysulfosuccinimide (Sulfo-NHS, Thermo Scientific, US) at aconcentration of 125 mM were added to the solution, and were then mixedby stirring at room temperature for 15 minutes to prepare an activesolution in which cotinine-NHS ester was formed. A sodium hydroxidesolution was added to adjust the pH of the above-mentioned activesolution to 7 or more for optimal reaction between the cotinine-NHSester and the amine group of the protein, and 1 mL of this activesolution was mixed with the same volume of the antibody to be conjugatedwith cotinine at a concentration of 25 μM. The mixture was reacted bystirring at room temperature for 3 hours to obtain a cotinine-antibodyconjugates produced via an EDC coupling reaction. The cotinine-antibodyconjugates thus obtained was dialyzed with PBS using the SLIDE-A-LYZER™Dialysis Cassette (Thermo Fisher Scientific, US), or was subject toreplacing the buffer solution with PBS using the AMICON® UltraCentrifugal Filter (EMD Millipore, US).

3-2. Preparation of Complex of Cotinine and Aptamer

A cotinine-pegaptanib conjugate was prepared from pegaptanib, an aptamerthat recognizes the vascular endothelial growth factor (VEGF), which wasgenerated by the solid-phase oligopeptide synthesis method.

In detail, a pegaptanib RNA aptamer(5′-pCfpGmpGmpArpArpUfpCfpAmpGmpUfpGmpAmpAmpUfpGmpCfpUfpUfpAmpUfpAmpCfpAmpUfpCfpCfpGm-p-dT-3-3′, SEQ ID NO: 35) was synthesized fromthe 3′-terminus to the 5 ‘-terminus using an oligopeptide synthesizer,and an amino C6 linker was attached to the 5’-terminus. Chemicalconjugation with cotinine was performed using the active estercross-linking method for the amino C6 linker, and then purified (>95%purity) by reverse phase high pressure liquid chromatography using anXBRIDGE™ Prep C18 column (5 μm, 10×150 mm, Waters Corp., US). The massthereof was determined using a mass spectrometer (ST Pharm, Korea)(Clin. Chim. Acta., 2010(411):1238; Exp. Mol. Med., 2012(44):554).

The synthesized pegaptanib cotinine-conjugate suspended in distilledwater containing diethyl pyrocarbonate was denatured at 95° C. for 5minutes, cooled at room temperature for 30 minutes, and then stored at−20° C.

Comparative Example 1. Preparation of Chimeric Antigen Receptor T CellsLinked with Anti-Carcinoembryonic Antigen scFv Fragment

A chimeric antigen receptor gene linked with the anti-carcinoembryonicantigen (anti-CEA) scFv fragment was obtained by PCR method by using theprimers listed in Table 1 above using the pLxSN-scFv-anti-CEA-zetaplasmid (Dr. Philip Darcy, PeterMac Cancer Center, Australia) as atemplate. The obtained gene and the pMSCV-puro vector (K1062-1,Clontech, US) were cleaved with HindIII and ClaI respectively andligated together. The prepared construct was named pMP-C28Z. Using theconstruct, a retrovirus was prepared under the conditions and methods ofExamples 1 and 2, and T cells transduced with the retrovirus wereprepared.

Comparative Example 3. Preparation of T Cells Expressing GreenFluorescent Protein

In order to prepare T cells expressing the green fluorescent protein,the gene of the green fluorescent protein(GFP) included in the pMIG-wplasmid (Dr. Yosef Refaeli, National Jewish Medical and Research Center,US) was obtained by digestion of the plasmid with NcoI and SalI. Thedigested GFP gene fragment was inserted into the NcoI/SalI site of thepMP-C28Z plasmid prepared in Comparative Example 2 after removing theanti-carcinoembryonic chimeric antigen receptor gene that was includedin this plasmid. The prepared construct was named pMP-GFP. Afterwards,the construct was used to produce a retrovirus under the conditions andmethods of Examples 1 and 2, and T cells transduced with the retroviruswere produced.

Test Example 1. Confirmation of Activation of Chimeric Antigen ReceptorT Cells Linked with Anti-Cotinine Antibody Fragment UsingCotinine-Pegaptanib Conjugate

Using the cotinine-pegaptanib conjugate prepared in Example 3-2 above,whether the conjugate recognizes the vascular endothelial growth factor(VEGF) and thereby induces activation of chimeric antigen receptor cellswas examined.

First, 5 μg/mL of VEGF protein was added to a 96-well plate, and then,the plate was placed overnight at 4° C. for coating the plate with VEGF.The coated plate was washed twice with the RPMI-1640 medium (Welgene,Korea) supplemented with 20% fetal bovine serum and 1%penicillin-streptomycin (Gibco, US), and the 100-nM cotinine-pegaptanibconjugate was added and incubated at 37° C. for 1 hour. After thereaction, the plate was washed 3 times with the RPMI-1640 medium, andthen the chimeric antigen receptor T cells prepared in Example 2 wereadded so that there were 1×10⁵ cells per well, and the cells werecultured at 37° C. under 5% CO₂ for 24 hours. Then, the amount ofinterferon gamma secreted in the medium was measured according to themanufacturer's protocol using an ELISA kit (Cat #. 555138, BDBioscience, US), and the results are shown in FIG. 4 . As controlgroups, a group (VEGF only) in which nothing was added to a plate coatedwith VEGF, a group (T cell only) in which only T cells were added to aplate not coated with VEGF, a group (VEGF+T cell) in which only T cellswere added to a plate coated with VEGF, and a group (VEGF+ControlAptamer+T cell) in which a control aptamer(5′-dTTGGTGGTGGTGGTTGTGGTGGTGGTGG-3′, SEQ ID NO: 36) and T cells wereadded to a plate coated with VEGF, were used.

As shown in FIG. 4 , it was confirmed that secretion of interferon gammawas significantly increased in the group (VEGF+Cot-pegaptanib+T cell) inwhich the cotinine-pegaptanib conjugate was added to the chimericantigen receptor T cells linked with the anti-cotinine antibodyfragment.

Test Example 2. Confirmation of Activation of Chimeric Antigen ReceptorT Cells Linked with Anti-Cotinine Antibody Fragment UsingCotinine-Anti-HER2 Antibody Conjugate

Using the cotinine-anti-HER2 antibody conjugate prepared in Example 3-1,it was determined whether the conjugate induces activation of chimericantigen receptor cells by recognizing HER2 present on cell surfaces.

First, the AU565 cell line, which is an HER2 positive cell, and theMDA-MB-231 cell line, which is an HER2 negative cell, were dispensed inwells of 96-well round bottom plate so that there were 3×10⁴ cells perwell, and were cultured overnight under conditions of 5% CO₂ and 37° C.After removal of the culture supernatant, 100 μL of thecotinine-anti-HER2 antibody conjugate added to a concentration 10 μg/mLin the DMEM medium (Gibco, US) supplemented with 10% fetal bovine serumand 1% penicillin-streptomycin was added, and was cultured under thesame conditions for 1 hour. Thereafter, the medium was removed, thecells were washed twice with a serum-free medium, and then the chimericantigen receptor T cells prepared in Example 2 were added so that therewere 3×10⁵ cells per well, and cultured for 24 hours under conditions of5% CO² and 37° C. Then, the amount of interferon gamma secreted in themedium was measured according to the manufacturer's protocol using anELISA kit, and the results are shown in FIG. 5 . As control groups, agroup (T cell only) in which chimeric antigen receptor T cells wereadded to a plate in which tumor cells were not cultured, a group (tumoronly) in which nothing was added to a plate in which tumor cells werecultured, a group (tumor+T cell) in which chimeric antigen receptor Tcells were added to a plate in which tumor cells were cultured, a group(CT302) in which an influenza virus hemagglutinin-specific antibody(CT302) and chimeric antigen receptor T cells were added to a plate inwhich cells were cultured, a group (CT302-Cot) in whichcotinine-conjugated CT302 antibody and chimeric receptor T cells wereadded to a plate in which tumor cells were cultured, and a group(anti-HER2) in which the free anti-HER2 antibody and chimeric antigenreceptor T cells were added to a plate in which cells were cultured,were used.

As shown in FIG. 5 , it was confirmed that secretion of interferon gammawas significantly increased in the group (anti-HER2-Cot) in which thecotinine-anti-HER2 antibody conjugate was added to the chimeric antigenreceptor T cells linked with the anti-cotinine antibody fragment.

Test Example 3. Confirmation of Antigen-Specific Activation of ChimericAntigen Receptor T Cells Linked with Anti-Cotinine Antibody Fragment

In order to determine whether secretion of interferon gamma induced bychimeric antigen receptor T cells linked with the anti-cotinine antibodyfragment complexed with the cotinine-anti-HER2 antibody conjugate isspecific to the antibody linked with cotinine, the following experimentwas carried out.

The experiment was carried out under the same conditions and methods asin Test Example 2, except that only AU565 cells which were HER2 positivecells were used. T cells not expressing anything on the surface, T cellsprepared in Comparative Example 1, or T cells prepared in Example 2 wereeach added in combination with a group (T cell only) in which only Tcells were added to a plate in which tumor cells were not cultured, agroup (CT302) in which the CT302 antibody and T cells were added to aplate in which tumor cells were cultured, a group (CT302-Cot) in whichcotinine conjugated with the CT302 antibody and T cells were added to aplate in which tumor cells were cultured, a group (anti-HER2) in whichthe free anti-HER2 antibody and T cells were added to a plate in whichcells were cultured, and a group (anti-HER2-Cot) in which cotinineconjugated with the anti-HER2 antibody and T cells were added to a platein which tumor cells were cultured. As a result, the amount of secretedinterferon gamma was measured by the ELISA method and shown in FIG. 6A.

As shown in FIG. 6A, it was confirmed that secretion of interferon gammawas significantly increased in the group (anti-HER2-Cot) in which thecotinine-anti-HER2 antibody conjugate was added to chimeric antigenreceptor T cells linked with the anti-cotinine antibody fragment.Therefore, it was found that the secretion of interferon gamma wasspecifically induced by the binding molecule conjugated with cotinine.

Test Example 4. Confirmation of Expression of Chimeric Antigen ReceptorPresented on Surface of T Cells

The expression of the chimeric antigen receptor was determined bymeasuring the positivity of c-myc tag staining in T cells that do notexpress any chimeric antigen receptor on the surface and the chimericantigen receptor T cells linked with the anti-cotinine antibody fragmentor with the anti-CEA antibody fragement, prepared in Example 2 andComparative Example 1 respectively.

First, T cells that do not express any chimeric antigen receptor on thesurface and the chimeric antigen receptor T cells prepared in Example 2or Comparative Example 1 were dispensed so that there were 4×10⁵ cellsper well. The dispensed cells were washed twice with a PBS buffersolution supplemented with 1% bovine serum albumin (BSA) and 0.02% NaN3,and the anti-c-myc antibody (BD Bioscience, US) was added after dilutingat a ratio of 1:400 in the buffer solution. The mixtures were reactedfor 20 minutes under refrigeration and washed 3 times with the PBSbuffer solution. After adding 50 μL of a PBS supplemented withstreptavidin-PE (BD Bioscience, US) at a ratio of 1:1000,anti-CD8-perCP-Cy5.5 (BD Bioscience, US) at a ratio of 1:500, andanti-CD4-APC (BD Bioscience, US) at a ratio of 1:1000, the mixtures werereacted for 25 minutes under refrigeration. After being washed threetimes with the PBS buffer, the cells were resuspended in a total of 300μL of PBS. c-myc positive cells were analyzed using a flow cytometer(FACS Caliber, BD Bioscience, US), and the results are shown in FIG. 6B.A group (unstained) treated with no antibody and a group (control)treated with only streptavidin-PE, which is a secondary antibody, wereused as control groups for the T cells.

As shown in FIG. 6B, it was confirmed that the chimeric antigen receptorT cells prepared in Example 2 and Comparative Example 1 expressed theanti-cotinine antibody fragment or anti-CEA antibody fragment.

Test Example 5. Confirmation of Activation of Chimeric Antigen ReceptorT Cells Linked with Anti-Cotinine Antibody Fragment UsingCotinine-Anti-CD20 Antibody Conjugate

Using the cotinine-anti-CD20 antibody conjugate prepared in Example 3-1,it was determined whether the conjugate induces activation of chimericantigen receptor cells by recognizing CD20 on the cell surface.Specifically, an experiment was carried out under the same conditionsand methods as in Test Example 2, except that instead of the AU565 cellline and the MDA-MB-231 cell line, a Jurkat E6.1 cell line, which is aCD20 negative cell, and a Raji cell line, which is a CD20 positive cell,were cultured in the RPMI-1640 medium supplemented with 10% fetal bovineserum and 1% penicillin-streptomycin, and the amount of secretedinterferon gamma was determined in a culture solution obtained after 48hours of culturing. As a result, the amounts of secreted interferongamma are shown in FIG. 7 . A group (T cell only) in which chimericantigen receptor T cells were added to a plate in which cells were notcultured, a group (tumor only) in which nothing was added to a plate inwhich tumor cells were cultured, and a group (anti-CD20) in which theanti-CD20 antibody and chimeric antigen receptor T cells were added to aplate in which cells were cultured were used as control groups.

As shown in FIG. 7 , it was confirmed that secretion of interferon gammawas significantly increased in a group (anti-CD20-Cot) in which thecotinine-anti-CD20 antibody conjugate was added to the chimeric antigenreceptor T cells linked with the anti-cotinine antibody fragment.

Test Example 6. Confirmation of Activation of Chimeric Antigen ReceptorT Cells Linked with Anti-Cotinine Antibody Fragment UsingCotinine-Anti-HLA Antibody Conjugate

Using the cotinine-anti-HLA antibody conjugate prepared in Example 3-1,it was determined whether the conjugate induces activation of chimericantigen receptor cells by recognizing HLA on the cell surface.Specifically, the experiment was carried out under the same conditionsand methods as in Test Example 2, except that instead of the AU565 cellline and the MDA-MB-231 cell line, an NIH3T3 cell line, which is an HLAnegative cell, and an HLA7 or HLA20 cell line, which is an HLA positivecell, were cultured in the IMDM medium supplemented with 20% fetalbovine serum and 1% penicillin-streptomycin, and the amount of secretedinterferon gamma was determined in a culture solution obtained after 48hours of culturing.

Meanwhile, the HLA7 and HLA20 cell lines were prepared by the followingmethod. Specifically, a mixed solution of 10 mL of peripheral blood andthe same amount of PBS was dispensed to 15 mL of FICOLL-PAQUE™ PLUS (GEHealthcare Life Science, US) and centrifuged at 400×g for 30 minutes,with the brake set to “0”. After centrifugation, the plasma in theuppermost layer was removed, and 5 mL of a peripheral blood mononuclearcell layer was taken. 30 mL of the RPMI-1640 medium was added thereto,and centrifugation was performed for 10 minutes under the sameconditions. The supernatant was removed, and the same amount of theRPMI-1640 medium was added to perform another wash. The precipitatedcells were resuspended in 2 mL of the RPMI-1640 medium including 20%fetal bovine serum, and the number of cells was counted. 1×10⁷ cellswere dispensed into a T25 flask, and a total of 6 mL of a culture mediumwas added and cultured under conditions of 5% CO₂ and 37° C. 2 mL ofEpstein-Barr virus (VR-1492, ATCC, US) was added thereto and culturedunder the same conditions. After two hours of culturing, 1 μg/mL ofcyclosporin A (Gibco, US) was added and cultured under the sameconditions for 4 days. On the 4th day of culturing, 2 mL of a culturemedium was added, and on the 7th day, was subcultured. When B cellstransduced with Epstein-Barr virus formed colonies for 10 to 20 days bycontinuing the subculture, two colonies were taken to continuesubculturing. The two immortalized B cell lines thus prepared were namedHLA7 and HLA20, respectively.

As a result, the amounts of secreted interferon gamma are shown in FIG.8 . A group (T cell only) in which chimeric antigen receptor T cellswere added to a plate in which tumor cells were not cultured, a group(tumor only) in which nothing was added to a plate in which tumor cellswere cultured, and a group (anti-HLA) in which the free anti-HLAantibody and chimeric antigen receptor T cells were added to a plate inwhich cells were cultured were used as control groups.

As shown in FIG. 8 , it was confirmed that secretion of interferon gammawas significantly increased in a group (anti-HLA-Cot) in which thecotinine-anti-HLA antibody conjugate was added to the chimeric antigenreceptor T cells linked with the anti-cotinine antibody fragment.

Test Example 7. Confirmation of Target Cell-Specific Apoptotic Effect byChimeric Antigen Receptor T Cells Linked with Cotinine-Anti-HER2Antibody Conjugate and Anti-Cotinine Antibody Fragment

Using the chimeric antigen receptor T cells linked with theanti-cotinine antibody fragment and the cotinine-anti-HER2 antibodyconjugate prepared in Example 3-1, it was determined whether thechimeric antigen receptor T cells induces cell death of the HER-positivetumor cells by recognizing HER2 on the cell surface.

First, the AU565 cell line, which is an HER2 positive cell, andMDA-MB-231 cell line, which is an HER2 negative cell, were dispensedsuch that 2×10⁶ cells were in a 100 mm cell culture container. Thedispensed cells were cultured under the same medium and the conditionsas in Test Example 2. The cultured cells were counted, and the cellswere prepared so that there were 2×10⁷ cells per 1 mL for the AU565 cellline and 2×10⁷ cells per 1 mL for the MDA-MB-231 cell line, andcarboxyfluorescein succinimidyl ester (CF SE, Invitrogen, US) was addedthereto to a concentration of 1 μM and 100 nM, respectively. Afterreacting at room temperature for 8 minutes so that each cell line wasstained to different fluorescence intensities, the results thereof weredetermined using a flow cytometer (BD Bioscience, US) and shown in FIG.9A.

As shown in FIG. 9A, the AU565 cell line was stained to a weakfluorescence intensity (CFSE^(low)), and the MDA-MB-231 cell line wasstained to a strong fluorescence intensity (CFSE^(high)).

The stained cells were washed twice with PBS, and the washed cell lineswere mixed in 1:1 ratio of 1.5×10⁶ cells each and dispensed into a96-well plate. When the dispensed cells were adhered, thecotinine-anti-HER2 antibody conjugate at 1 μg/mL was added thereto andallowed to react at room temperature for 1 hour. After the reaction, thecell lines were washed with a culture medium, counted, and placed in a5-mL tube so that there were 5×10⁴ cells. Herein, the chimeric antigenreceptor T cells linked with the anti-cotinine antibody fragment wereadded and cultured for 4 hours or 22 hours under conditions of 5% CO₂and 37° C. Here, the ratio of the chimeric antigen receptor T cells(working cell line, E), and the AU565 and MDA-MB-231 cell lines (targetcell line, T) was set to be 0:1, 5:1, or 10:1. After culturing, in orderto distinguish dead cells, 0.25 μg of 7-AAD (BD Bioscience, US) wasadded per 1×10⁶ cells and reacted at room temperature for 10 minutes,and then was analyzed by a flow cytometer. Here, 7-AAD negative cellsand CFSE positive cells were fractionated and analyzed. As a result ofthe analysis, the rate of cell death of the target cell was calculatedaccording to Equation 1 below, and the result is shown in FIG. 9B.

$\begin{matrix}{{{Rate}{of}{Apoptosis}} = {100 - \lbrack {\frac{\frac{\begin{matrix}{{Ratio}{of}{AU}565{cells}{in}{target}{cell}{line}} \\{{cultured}{with}{working}t{cells}}\end{matrix}}{\begin{matrix}{{{Ratio}{of}{MDA}} - {MB} - {231{cells}{in}{target}}} \\{{cell}{line}{cultured}{with}{working}T{cells}}\end{matrix}}}{\frac{\begin{matrix}{{Ratio}{of}{AU}565{cells}{in}{target}{cell}{line}} \\{{cultured}{without}{working}t{cells}}\end{matrix}}{\begin{matrix}{{{Ratio}{of}{MDA}} - {MB} - {231{cells}{in}{target}}} \\{{cell}{line}{cultured}{without}{working}T{cells}}\end{matrix}}} \times 100} \rbrack}} & \lbrack {{Equation}1} \rbrack\end{matrix}$

As shown in FIG. 9B, it was confirmed that cell death of AU565, which isan HER2 positive cell line, increased in proportion to the amount of theadded chimeric antigen receptor T cells. Thus, it was found that thechimeric antigen receptor T cells linked with the anti-cotinine antibodyfragment induces cell death specifically to the binding moleculeconjugated with cotinine.

Test Example 8. Confirmation of Cell Death-Inducing Effect of ChimericAntigen Receptor T Cells by Cotinine-Cytotoxic Agent Conjugate

A cotinine-cytotoxic agent conjugate was prepared by ConcortisBiotherapeutics, and it was determined whether the conjugate inducescell death of the chimeric antigen receptor T cells prepared in Example2 by the following method.

Specifically, the chimeric antigen receptor T cells were dispensed in a96-well plate so that there were 5×10⁵ cells and cultured in a culturemedium. A 0, 0.1, 1, 10, 100, or 1000 nM cotinine-cytotoxic agentconjugate was added thereto and cultured for 48 hours under conditionsof 5% CO₂ and 37° C. For the added cotinine-cytotoxic agent conjugate, acotinine-duocarmycin single conjugate, a cotinine-DM1 complex conjugate(2×cotinine-4×DM 1), or cotinine-duocarmycin complex conjugate(2×cotinine-4×duocarmycin) was used (FIG. 11 ). After culturing, livingcells were stained with 7-AAD, and cells stained with 7-AAD or withc-myc tag were analyzed by a flow cytometer. As a result of theanalysis, the cytotoxicity of the chimeric antigen receptor T cells dueto the cotinine-cytotoxic agent conjugate was calculated using Equation2 below, and the results are shown in FIGS. 10A, 10B and 10C. The Tcells prepared in Comparative Example 2 were used as a control group.

$\begin{matrix}{{{Relative}{Viability}(\%)} = {\frac{\begin{matrix}{{Positive}{rate}{of}{myc}{staining}{in}{group}{with}} \\{{conjugated}{administration} \times {Number}{of}{surviving}} \\{{cells}{in}{group}{with}{conjugate}{administration}}\end{matrix}}{\begin{matrix}{{Positive}{rate}{of}{myc}{staining}{in}{group}{without}} \\{{conjugated}{administration} \times {Number}{of}{surviving}} \\{{cells}{in}{group}{without}{conjugate}{administration}}\end{matrix}} \times 100}} & \lbrack {{Equation}2} \rbrack\end{matrix}$

As shown in FIGS. 10A, 10B and 10C, it was confirmed that much lowerdoses of cotinine-cytotoxic agent conjugate can induce cell death of thechimeric antigen receptor T cells linked with the anti-cotinine antibodyfragment compared to the T cells of Comparative Example 2.

From the above results, it was found that using the small amount ofcotinine-cytotoxic agent conjugate, cell death of the chimeric antigenreceptor T cells linked with the anti-cotinine antibody fragment can beinduced.

1. A method for inducing cell death of a chimeric antigen receptor cellin a subject in need thereof, the method comprising: administering acotinine-conjugated cytotoxic agent to the subject, wherein the subjectcarries the chimeric antigen receptor cell which is formed in thesubject by way of a previous administration of a cotinine-conjugatedbinding molecule and a cell transduced with a virus comprising a nucleicacid molecule, wherein the nucleic acid molecule encodes a chimericantigen receptor which is linked with an anti-cotinine antibody, andwherein the chimeric antigen receptor comprises: an anti-cotinineantibody or a fragment thereof; a hinge domain; a transmembrane domain;and a signal transduction domain.
 2. The method of claim 1, wherein thecytotoxic agent is duocarmycin, SN-38, calicheamicin, monomethylauristatin E (MMAE), monomethyl auristatin F (MMAF), doxorubicin,pyrrolobenzodiazepine, DM4, or DM1.
 3. The method of claim 1, whereinthe cell transduced with a virus is a T cell, a natural killer cell, ora macrophage.
 4. The method of claim 1, wherein the chimeric antigenreceptor comprises, in the order from the N-terminus to the C-terminusof the chimeric antigen receptor, the following (a)-(d): (a) ananti-cotinine single chain Fv (scFv) antibody, wherein the anti-cotininescFv antibody comprises the amino acid sequence of SEQ ID NO: 1; (b) aCD8 (cluster of differentiation 8) hinge domain comprising the sequenceof SEQ ID NO: 3; (c) a transmembrane domain of CD28 comprising thesequence of SEQ ID NO: 5 or 7; and (d) a signal transduction domainselected from the group consisting of the following (i)-(iii): (i) aCD28 cytoplasmic region comprising the sequence of SEQ ID NO: 9 or 11,(ii) a CD3 zeta cytoplasmic region comprising the sequence of SEQ ID NO:13, and (iii) a CD28 cytoplasmic region comprising the sequence of SEQID NO: 9 or 11 and a CD3 zeta cytoplasmic region comprising the sequenceof SEQ ID NO:
 13. 5. The method of claim 1, wherein the signaltransduction domain is the (iii) CD28 cytoplasmic region comprising thesequence of SEQ ID NO: 9 or 11 and CD3 zeta cytoplasmic regioncomprising the sequence of SEQ ID NO:
 13. 6. The method of claim 1,wherein the chimeric antigen receptor comprises the amino acid sequenceof SEQ ID NO: 15 or SEQ ID NO: 17.